A stability-indicating HPLC assay with diode array detection for the determination of a benzylpenicillin prodrug in aqueous solutions.

The aim of this study was to develop a stability-indicating HPLC assay for the determination of penethamate (PNT), an ester prodrug of benzylpenicillin (BP), in aqueous solutions. The method was validated by subjecting PNT to forced decomposition under stress conditions of acid, alkali, water hydrolysis and oxidation. A quenching solution was developed to limit degradation to negligible levels before and during the analysis. Both PNT and BP were simultaneously determined and separated in presence of degradation products on a C(18) column using a mobile phase consisting of methanol-acetonitrile-acetate buffer. Different degradation products were formed in the stress conditions. The peak purity indexes of PNT and BP obtained by diode array detection were >0.999, confirming the absence of other co-eluting substances. The assay was linear for both analytes in the concentration range 1-100 microg mL(-1). The LOD and LOQ of PNT were 0.03 and 0.09 microg mL(-1) respectively. Degradation of PNT followed pseudo-first-order kinetics with t(1/2) of 43.6 min at pH 2.01 and 4.2 min at pH 9.31. In addition, the absence of BP in the acidic solutions of PNT emphasises the futility of monitoring BP to assess the stability of PNT. In conclusion, the assay is rapid and stability-indicating with adequate precision and accuracy, and in conjunction with the quenching solution, can be used for stability studies of PNT with simultaneous quantitation of BP. The degradation studies provide useful information for formulation development of PNT.